Feb 21, 2022
Version 3
Dephosphorylation of 5´-ends of DNA using CIP (M0290) V.3
- 1New England Biolabs
Protocol Citation: New England Biolabs 2022. Dephosphorylation of 5´-ends of DNA using CIP (M0290). protocols.io https://dx.doi.org/10.17504/protocols.io.bddbi22nVersion created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 07, 2020
Last Modified: February 21, 2022
Protocol Integer ID: 33923
Keywords: Dephosphorylation, CIP, Calf intestine phosphatase, DNA,
Abstract
Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction. Uses the Calf Intestinal Alkaline Phosphatase (CIP - M0290).
Guidelines
Dephosphorylation of 5' -ends of DNA in Restriction Enzyme Reaction
- The phosphate can be added directly into the digestion reaction during or after DNA digestion
- CIP is active in all NEB restriction enzyme buffers
- DNA purification is required before ligation
Materials
MATERIALS
Alkaline Phosphatase, Calf Intestinal (CIP)New England BiolabsCatalog #M0290
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Prepare a 20 µL reaction as follows:
| A | B | |
| COMPONENT | AMOUNT | |
| DNA | 1 pmol of DNA ends* | |
| CutSmart® Buffer (10X) | 2 μl | |
| CIP | 1 unit | |
| H2O, purified | to 20 μl** |
* Note: 1 pmol of DNA ends is about 1 μg of a 3 kb plasmid.
** Scale larger reaction volumes proportionally.
Incubate at 37 °C for 00:30:00 .